Stress response induced by carbon nanoparticles in Paracentrotus lividus.

Members of the 14-3-3 protein family are involved in many important cellular events, including stress response, survival and apoptosis. Genes of the 14-3-3 family are conserved from plants to humans, and some members are responsive to UV radiation. Despite the high rate of pollution generated by nano-pollutants, up to now their toxic effect on development is totally obscure. Embryos treated with carbon nanoparticles, RNA preparation, retro-transcription and quantitative real-time PCR. In response to carbon nano-particles exposure, the embryos collected 24 h later showed a 3,07-fold at 5x10(12) p and a 1,58-fold at 2.5x10(13) p and a 1,92-fold at 2.5x10(14) p increase in Pl14-3-3ε transcript levels compared with controls. The Pl14-3-3ε mRNA delocalization parallels the failure in archenteron elongation observed morphologically, as well as the lack of specific endoderm markers. Here, we report the isolation of the complete cDNA encoding the 14-3-3 epsilon isoform from Paracentrotus lividus sea urchin embryos, referred to as Pl14-3-3ε. Pl14-3-3ε mRNA levels were measured by RT-PCR during development and found to increase from the mesenchyme blastula to the prism stage. Our results confirm the involvement of 14-3-3ε in the stress response elicited by carbon nano-particles.

The term nano-pollution is generically referring to all waste generated during the fabrication of nanomaterials.These types of waste can be very dangerous because of their size, nanoparticles in the free state can be released into the air during production (or production accidents) or as waste, only to accumulate in the soil, in water or vegetable and thus be assimilated by the animals causing unknown effects.An environmental assessment of this phenomenon is very important because it could lead to define new environmental impacts.Currently you can not accurately predict or control the ecological impacts due to the release of these nano-pollutants in the environment.
Ecotoxicological impacts of nanoparticles and the potential for bioaccumulation in plants and microorganisms are still under research.
In this regard, this work has examined the sea urchin Paracentrotus lividus, a model organism used in studies on embryonic development, Downloaded from ijmcmed.org at 8:00 +0430 on Thursday April 9th 2020 and was exposed to the effects of carbon nanoparticles, synthesized according to the electrochemical method.
Members of the 14-3-3 protein family are involved in many important cellular events, including stress response, survival and apoptosis.
Genes of the 14-3-3 family are conserved from plants to humans, and some members are responsive to UV radiation.Despite the high rate of pollution generated by nano-pollutants, up to now is totally obscure their toxic effect on development.
Proteins of the 14-3-3 family are considered chaperones/adaptors, playing important roles in cellular homeostasis (2,3).Their involvement in stress response has been claimed as evidenced by the increased protein/mRNA levels following exposure to drugs and pesticides.For example, different subsets of 14-3-3 genes were induced after treatment with the fungal toxin fusicoccin in the tomato plant, demonstrating a correlation with the pathogen-associated defense response (4).In sponges, the 14-3-3 gene is induced by pesticides (PCB 118), in parallel with the increase in the levels of the heat shock protein 70 transcript, suggesting their role in preventing apoptosis (5).
Under physiological conditions 14-3-3 homo-and hetero-dimers (6,7) can interact with a wide variety of signalling proteins, including the stress signalling BAD/BAX mitochondrial proteins and the FOXO transcription factor.This interaction is possible only if 14-3-3 proteins are unphosphorylated and, by sequestering BAD, BAX and FOXO in the cytoplasm, their entrance into the mitochondrion/nucleus is prevented.
At least one isoform has been found in the sponge Geodia cynodium (5), and in the sea urchins Heliocidaris tuberculata, Heliocidaris erythrogramma (14).Three isoforms were annotated in the genome of Strongylocentrotus purpuratus, one referred to as ε, and two known as the isoforms 1 and 2 (15,16).
The sea urchin embryo is one of the most used marine invertebrates models for studying apoptosis, cellular stress and biochemical markers of pollution (17)(18)(19)(20)(21)(22).It offers a suitable model for toxicological and developmental studies as the feeding larva (pluteus) is complete in about 48 h; the specification of the embryonic territories, including ectoderm, mesoderm and endoderm, begins as early as the 32-cells cleavage stage (23).
The aim of this study was to investigate the temporal and spatial expression of the 14-3-3ε gene in P. lividus sea urchin embryos, during

Embryo culture and micromanipulation
Adult Paracentrotus lividus sea urchins were collected along the coast of Salento.Eggs were fertilized and embryos cultured at a dilution of 4.000/mL in Millipore Filtered Sea Water (MFSW) containing antibiotics, at 16-18°C.Animals were induced to shed gametes by intracoelomic injection of 0.5 M KCl.Eggs were washed several times with filtered, natural seawater (SW), fertilized with a Downloaded from ijmcmed.org at 8:00 +0430 on Thursday April 9th 2020 dilute suspension of sperm and cultured in SW in glass bowls.

Morphological analysis
The morphological analysis of perturbed and control embryos was performed using an inverted microscope Nikon Eclipse 80i, the images were recorded by a digital camera Nikon DMX 1200F.
Embryos at different developmental stages (6h,24h) were collected by low-speed centrifugation.

RNA preparation and relative RT-PCR analysis
Total RNA was isolated according to the manufacturer's instructions using TRIzol® Plus RNA Purification System (Invitrogen) from 25 collected embryos, frozen in liquid nitrogen and stored at -80°C until use.Briefly, total RNA (20µg) isolated from control and embryos treated with carbon nano-particles as described above were run on 1.5% agarose gel, under denaturing conditions (formamide 50%, MOPS 1×, formaldeide 5.5%).

Quantitative real-time PCR
cDNAs were synthesized according to a single-step ThermoScript™ RT-PCR Systems kit (Invitrogen) protocol.About 0.1-1% of each RT reaction was used to run real-time PCR on a SmartCycler System (Cepheid) with SYBR ® Green JumpStart Taq ReadyMix (Sigma-Aldrich) and the primer pairs indicated in Table 1.Real-time PCR samples were run in triplicate.Quantification measurements of gene expression were performed as described by the manual of Applied Biosystems Step One Plus real time PCR, a Comparative Threshold Cycle Method, using SYBR Green chemistry (24).Pl-S24 mRNA was used as the internal endogenous reference gene (25).

Synthesis of Carbon nanoparticles
Carbon nanoparticles colloidal solution was obtained in the following.High purity graphite rod (SPI 99.99%) was used as an anode (5 mm diam), and a stainless steel rod (SWAGELOK AISI 1016,   measurements were performed using a comparative threshold cycle method, in which the Pl-S24 cDNA PCR product was used as an endogenous control.gene, which was assumed to be constant during development.

UV-Vis absorption measurements
The egg cDNA was used as reference sample and was assumed as 1 in arbitrary units.The This study has highlighted the negative effects of carbon nanoparticles in aquatic species and, therefore, we believe that we should pay particular attention to all applications involving the use of carbon nanomaterials.
In conclusion, we have demonstrated a direct relationship between carbon nanoparticles exposure of sea urchin embryos and Pl14-3-3ε mRNA levels, suggesting its implication in the regulative cascade activated in the stress response.To the best of our knowledge this is the first time that a carbon nanoparticles induced 14-3-3ε transcriptional regulative mechanism has been demonstrated.As previously described, upon stress 14-3-3 proteins are phosphorylated by the JNK kinase and are thus unable to bind BAD, BAX and FOXO, which are then able to translocate to mitochondria and nuclei, exerting their pro-apoptotic functions (31,32).The increase in Pl14-3-3ε mRNA detected after NPC stress could result in higher 14-3-3 protein levels (possibly not phosphorylated) which could then bind a large quantity of the above mentioned factors, determining cellular survival.
On the basis of studies described by Russo et al 2010 we confirmed the use of sea urchin embryos for examining the role of 14-3-3 in cell stress response pathways.Finally, 14-3-3ε could be used as a valuable molecular biomarker to identify the dangerous effects of sunlight occurring in marine organisms living in shallow waters.Previous studies showed a rapid induction of 14-3-3's in tomato plant roots deprived of iron (33).This is particularly interesting due to the close coupling of copper and iron metabolism (34), demonstrated by their shared cell surface reductases Fre1 and Fre2.Direct evidence for the role of 14-3-3 in stress response has been shown in the macro-alga Fucus vesiculosus (35).
development and in response to carbon nanoparticles treatment.The complete cDNA encoding Pl14-3-3ε was isolated by reverse transcriptase polymerase chain reaction (RT-PCR) from NPC exposed embryos mRNA.Quantitative real-time PCR (QPCR) analysis was used to assess the extent of gene expression.

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Fig 1. UV-vis absorption spectra of carbon nanoparticles immediately after the synthesis, after 72 and 144 hours.Related Raman spectrum (inset) A typical UV-Vis spectra set recorded in the range between 200 and 800 nm immediately after the synthesis and after 72 e 144 hours is shown in Fig 1 It is evident the presence of a well defined peak at 236 nm and a shoulder at ~310 nm, that can be attributed to π→π* transitions of aromatic C-C bonds and n→π* transitions of C=O bonds, respectively, according to the experimental data reported for the unreduced nanocarbon dispersion (26 27).It is interesting to note that the position of the peak remains unchanged over time, showing the stability of the nanoparticles dispersed in solution with a negligible aggregation and a sedimentation almost nothing.In the inset of Fig 1 is reported the Raman spectrum.The peaks at about 1350, 1582, 1617 and 2700 cm -1 are assignable to carbon-based materials in low-dimensional configuration sp2 that is nanographite particles having dimensions of a few nanometers (28).To evaluate the exact size and structure of nanoparticles and to understand the aggregation process and the mechanism involved (29) a morphological analysis has been performed by Transmission Electron Microscopy (TEM).A typical TEM image is reported in Fig 2, it is evident that the carbon nanoparticles were almost spherical and were great 3 nm with a statistical standard deviation (σ) of 2 nm as evident in the inset that report the histogram showing the size dispersion of observed carbon nanoparticles.Differential expression of Paracentrotus lividus 14-3-3ε mRNA in control embryos We performed quantitative PCR experiments with cDNA samples obtained by reverse transcription from total RNA extracted at different developmental stages of sea urchin embryos: unfertilized eggs as T0, T6 and T24.Quantitative Downloaded from ijmcmed.org at 8:00 +0430 on Thursday April 9th 2020

Fig 2 .
Fig 2. Transmission electron microscope image and histogram of silver nanoparticles size distribution (inset)

Fig 5 .
Fig 5.  RT-PCR of the Pl-14-3-3 epsilon gene in embryos incubated at 24 hours without and with the presence of carbon nanoparticles.
Downloaded from ijmcmed.org at 8:00 +0430 on Thursday April 9th 2020 Conclusion In this study, we observed the transcriptional response of gene 14-3-3 in Paracentrotus lividus, and provide data to demonstrate its response to carbon nanoparticles showing that a clear correlation exists between nanoparticles exposure and the transcriptional regulation of this gene.